This invention relates to arylsulfide, arylsulfoxide and arylsulfone derivatives which are xcex23 adrenergic receptor agonists useful for the treatment of metabolic disorders related to insulin resistance or hyperglycemia (typically associated with obesity or glucose intolerance), atherosclerosis, gastrointestinal disorders, neurogenetic inflammation, glaucoma, ocular hypertension, and and frequent urination, and are particularly useful in the treatment or inhibition of type II diabetes.
The subdivision of xcex2 adrenergic receptors (xcex2-AR) into xcex21- and xcex22-AR has led to the development of xcex21- and xcex22- antagonists and/or agonists which have been useful for the treatment of cardiovascular disease and asthma. The recent discovery of xe2x80x9catypicalxe2x80x9d receptors, later called xcex23-AR, has led to the development of xcex23-AR agnoists which may be potentially useful as antiobesity and antidiabetic agents. For recent reviews on xcex23-AR agnoists , see: 1. A. D. Strosberg, Annu. Rev. Pharmacol. Toxicol. 1997, 37, 421; 2. A. E. Weber, Ann. Rep. Med. Chem. 1998, 33, 193; 3. C. P. Kordik and A. B. Reitz, J. Med. Chem. 1999, 42, 181; 4. C. Weyer, J. F. Gautier and E. Danforth, Diabetes and Metabolism, 1999, 25, 11.
Compounds that are potent and selective xcex23 agonists, may be potentially useful antiobesity agents. Low levels or lack of xcex21 and xcex22-agonistic properties will minimize or eliminate the adverse side effects that are associated with xcex21 and xcex22 agonistic activities, i.e. increased heart rate, and muscle tremor, respectively.
Early developments in the xcex23-agonist field are described in European patent 427480, U.S. Pat. Nos. 4,396,627, 4,478,849, 4,999,377, 5,153,210. Although the early developments purport to claim compounds with greater xcex23-AR selectivity over the xcex21- and xcex22-AR. However, clinical trials in humans with those early developed xcex23-agonists have, so far, not been successful.
More recently, potent and selective human xcex23 agonists have been described in several patents and published applications: WO 98/32753, WO 97/46556, WO 97/37646, WO 97/15549, WO 97/25311, WO 96/16938, WO 95/29159, European Patents 659737, 801060, 714883, 764640, 827746, and U.S. Pat. Nos. 5,561,142, 5,705,515, 5,436,257, and 5578620. These compounds were evaluated in Chinese hamster ovary (CHO) cells test procedures, expressing cloned human xcex23 receptors, which predict the effects that can be expected in humans (Granneman et al., Mol Pharmacol., 1992, 42, 964; Emorine et al., Science, 1989, 245, 1118; Liggett Mol. Pharmacol., 1992, 42, 634).
xcex23-Adrenergic agonists also are useful in controlling the frequent urge of urination. It has been known that relaxation of the bladder detrusor is under beta adrenergic control (Li J H, Yasay G D and Kau S T Pharmacology 1992; 44: 13-18). Beta-adrenoceptor subtypes are in the detrusor of guinea-pig urinary bladder. Recently, a number of laboratories have provided experimental evidence of xcex23 adrenergic receptors in a number of animal species including human (Yamazaki Y, Takeda H, Akahane M, Igawa Y, et al. Br. J. Pharmacol. 1998; 124: 593-599), and that activation of the xcex23 receptor subtype by norepinephrine is responsible for relaxation of the urinary bladder.
Urge urinary incontinence is characterized by abnormal spontaneous bladder contractions that can be unrelated to bladder urine volume. Urge urinary incontinence is often referred to hyperactive or unstable bladder. Several etiologies exist and fall into two major categories, myogenic and neurogenic. The myogenic bladder is usually associated with detrusor hypertrophy secondary to bladder outlet obstruction, or with chronic urinary tract infection. Neurogenic bladders are associated with an uninhibited micturition reflex. An upper motor neuron disease is usually the underlying cause. In either case, the disease is characterized my abnormal spontaneous contractions that result in an abnormal sense of urinary urgency and involuntary urine loss. At present, the most common therapy for hyperactive bladder includes the use of antimuscarinic agents to block the action of the excitatory neurotransmitter acetylcholine. While effective in neurogenic bladders, their utility in myogenic bladders is questionable. In addition, due to severe dry mouth side-effects associated with antimuscarinic therapy, the patient compliance with these agents is only approximately 30%.
In the bladder, xcex23 adrenergic receptor agonists activate adenylyl cyclase and generate cAMP through the G-protein coupled xcex23 receptor. The resulting phosphorylation of phospholamban/calcium ATPase enhances uptake of calcium into the sarcoplasmic reticulum. The decrease in intracellular calcium inhibits bladder smooth muscle contractility.
It is suggested therefore, that activation of the xcex23 adrenergic receptor in the urinary bladder will inhibit abnormal spontaneous bladder contractions and be useful for the treatment of bladder hyperactivity. Note, that unlike the antimuscarinics, xcex23 adrenergic receptor agonists would be expected to be active against both neurogenic and myogenic etiologies.
Despite all these recent developments there is still no single therapy available for the treatment of type II diabetes (NIDDM), obesity, atherosclerosis, gastrointestinal disorders, neurogenetic inflammation, frequent urination and related diseases. A potent and selective xcex23 adrenergic receptor agonist is therefore highly desirable for the potential treatment of such disease states.
This invention provides compounds of Formula I having the structure 
wherein,
R1 is hydrogen, alkyl of 1-6 carbon atoms, halogen, trifluoromethyl, trifluoromethoxy, alkoxy of 1-6 carbon atoms, hydroxy, nitro, amino, cyano, carboxy, alkoxycarbonyl of 2-7 carbon atoms, aminocarbonyl, alkylamino of 1-6 carbon atoms, dialkylamino of 1-6 carbon atoms per alkyl group, acyl of 2-7 carbon atoms, acylamino of 2-7 carbon atoms, amino, phenylalkyl having 1-6 carbon atoms in the alkyl moiety, or xe2x80x94NR5SO2R5;
R2 is hydrogen, or alkyl of 1-6 carbon atoms;
R3 is hydrogen, alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, or halogen;
R4 is (a) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, S, and N, optionally substituted with R6; (b) a phenyl ring optionally substituted with R6; (c) phenylalkyl having 1-6 carbon atoms in the alkyl moiety; or (d) alkyl of 1-6 carbon atoms;
R5 is hydrogen, alkyl of 1-6 carbon atoms, or phenyl;
R6 is alkyl of 1-6 carbon atoms, halogen, trifluoromethyl, trifluoromethoxy, alkoxy of 1-6 carbon atoms, hydroxy, nitro, amino, cyano, aminocarbonyl, alkylamino of 1-6 carbon atoms, dialkylamino of 1-6 carbon atoms per alkyl group, acyl 2-7 carbon atoms, acymino of 2-7 carbon atoms, amirno, phenylalkyl having 1-6 carbon atoms in the alkyl moiety, xe2x80x94CO2R5, or xe2x80x94NR5SO2R5; or is a 5-6 heterocyclic ring having 1-4 heteroatoms selected from O, S, and N mono- or di- substituted with R2; 
is
(a) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S; or
(b) a phenyl ring; 
is
(a) a phenyl ring; or
(b) a phenyl fused to a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S;
Y is alkyl of 1-6 carbon atoms;
Z is a bond, or xe2x80x94OCH2xe2x80x94;
m is 1-2;
n is 0-2;
or a pharmaceutically acceptable salt thereof, which are selective agonists at human xcex23 adrenergic receptors and are useful in treating or inhibiting metabolic disorders related to insulin resistance or hyperglycemia (typically associated with obesity or glucose intolerance), atherosclerosis, gastrointestinal disorders, neurogenetic inflammation, glaucoma, ocular hypertension, and frequent urination; and are particularly useful in the treatment or inhibition of type II diabetes.
Pharmaceutically acceptable salts can be formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable aids when a compound of this invention contains a basic moiety. Salts may also be formed from organic and inorganic bases, such as alkali metal salts (for example, sodium, lithium, or potassium) alkaline earth metal salts, ammonium salts, alkylammonium salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon atoms in each alkyl group, and trialkylammonium salts containing 1-6 carbon atoms in each alkyl group, when a compound of this invention contains an acidic moiety.
Alkyl includes both straight chain as well as branched moieties. By definition alkyl also includes alkyl moieties which are optionally mono- or poly substituted with groups such as halogen, hydroxy, cyano, alkoxy, aryloxy, arylalkyl, alkylthio, arylthio, amino, alkylamino, and dialkylamino. Halogen means bromine, chlorine, fluorine, and iodine.
As used herein, a heterocyclic ring is a ring contining 1-4 heteroatoms selected from N, O, and S, which may be saturated, unstaurated, or partially unsaturated. It is understood that the heterocyclic ring does not contain heteroatoms in arrangements which would make them inherently unstable. For example, the term heterocyclic ring does not include ring systems containing Oxe2x80x94O bonds in the ring backbone. Preferred heterocycles and phenyl fused heterocycles include, but are not limited to, pyridyl, pyrimidinyl, pyrrolyl, piperidinyl, piperazinyl, thienyl, imidazolyl, thiazolyl, benzimidazolyl, benzotriazolyl, benzothiadiazolyl, oxadiazolyl, indolyl, benzofuranyl, dihydrobenzofuranyl, and methylenedioxyphenyl. More preferred heterocycles include oxadiazolyl, and isoxazolyl, and more preferred phenyl fused heterocycles include benzotriazolyl.
When B is a phenyl fused heterocycle, either ring of the phenyl fused heterocycle may be bonded to Z, and either ring may contain the R1 substitutent. When B contains more than one R1 group, they may be the same or different. Similarly, when R6 is xe2x80x94NR5SO2R5, the R5 substituents may be the same or different.
The compounds of the present invention contain at least one asymmetric center. Additional asymmetric centers may exist on the molecule depending upon the structure of the substituents on the molecule. The compounds may be prepared as a racemic mixture and can be used as such, or may be resolved into the. In addition to covering the racemic compounds, this invention also covers all individual isomers, enantiomers, diasteromers or mixtures thereof, regardless of whether the structural representations of the compounds indicate such stereochemistry.
Preferred compounds of Formula I are those in which
R1 is hydrogen, alkyl of 1-6 carbon atoms, halogen, trifluoromethoxy, alkoxy of 1-6 carbon atoms, hydroxy, cyano, or xe2x80x94NR5SO2R5;
R3 is hydrogen;
R6 is alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, or is a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, S, and N mono- or di- substituted with R2;
Y is methylene;
or a pharmaceutically acceptable salt thereof.
Specifically preferred compounds of this invention are:
a) 4-(4-{2-[(2S)-3-(1H-Benzotriazol-4-yloxy)-2-hydroxy-propylamino]-ethyl}-phenylsulfanylmethyl)-benzonitrile;
b) (2S)-1-(1H-Benzotriazol-4-yloxy)-3-{2-[4-(3-p-tolyl-[1,2,4]oxadiazol-5-ylmethylsulanyl)-phenyl]-ethylamino}-propan-2-ol;
c) 4-(3-{2-[4-(5-tert-Butyl-[1,2,4]oxadiazol-3-ylmethylsulfanyl)-phenyl]-ethylamino}-2-hydroxy-propoxy)-phenol;
d) 4-[(2S)-3-(2-{4-[5-(3,5-Dimethyl-isoxazol-4-yl)-[1,2,4]oxadiazol-3-ylmethanesulfonyl]-phenyl}-ethylamino)-2-hydroxy-propoxy]-phenol;
e) 4-((2S)-3-{2-[4-(5-tert-Butyl-[1,2,4]oxadiazol-3-ylmethanesulfonyl)-phenyl]-ethylamino}-2-hydroxy-propoxy)-phenol;
f) 4-[(2S)-2-Hydroxy-3-(2-{4-[5-(4-methoxy-phenyl)-[1,2,4]oxadiazol-3-ylmethylsulanyl]-phenyl}-ethylamino)-propoxy]-phenol;
g) 4-[(2S)-2-Hydroxy-3-(2-{4-[5-(4-methoxy-phenyl)-[1,2,4]oxadiazol-3-ylmethylsulanyl]-phenyl}-ethylamino)-propoxy]-2-methyl-phenol;
h) 4-((2S)-3-{2-[4-(5-Benzyl-[1,2,4]oxadiazol-3-ylmethanesulfinyl)-phenyl]-ethylamino}-2-hydroxy-propoxy)-phenol;
i) 2-[(4-{[(5-phenyl-1,2,4-oxadiazol-3-yl)methyl]sulfonyl}phenethyl)amino]-1-[4-(trifluoromethoxy)phenyl]-1-ethanol; and
j) N-[2-hydroxy-5-((1 R)-1-hydroxy-2-{[4-({[5-(4-methoxyphenyl)-1,2,4-oxadiazol-3-yl]methyl}sulfonyl)phenethyl]amino}ethyl)phenyl]methane sulfonamide
or a pharmaceutically acceptable salt thereof.
The compounds of this invention were be prepared according to the following schemes from commercially available starting materials or starting materials which can be prepared using literature procedures. These schemes show the preparation of representative compounds of this invention. The compounds of Formula(I) of the instant invention can be prepared from a reaction between epoxide intermediates, such as those of Formula (II), and amine intermediates, such as those of formula (III). The preparation of these intermediates is described in the following schemes. 
where n, Z, Y, A, B, R1, R2, R3, and R4, are as defined under Formula (I).
Compounds of Formula (II) are known in the literature or may be prepared by a variety of methods familiar to those skilled in the art of organic synthesis. One common route is illustrated in Scheme 1. Aldehyde 1, which is commercially available or prepared using methods available in the literature, is treated with sodium trimethylsulfoxonium ylide (made by treating trimethylsulfoxonium iodide with sodium hydride) in a solvent such as THF. The reaction mixture is processed and purified to afford the epoxide 2.
Another common route to compounds of Formula (II) is illustrated in Scheme 2. Phenol 3, which is commercially available or prepared using methods available in the literature, is treated with R(+)-glycidol, triphenylphosphine, and finally diethylazodicaboxylate. These conditions are commonly known, in the art, as Mitsunobu conditions. The reaction mixture is processed and purified to afford the epoxide 4. R1 substituents as well as others may need to be protected during the above or subsequent procedures. A description of the use of such protecting groups may be found in Protecting Groups in Organic Synthesis, 2nd Ed., T. W. Greene and P. G. M. Wuts, John Wiley and Sons, New York, 1991.
An alternative method that may be used to prepare compounds of Formula (II) is illustrated in Scheme 3. Phenol 3a, which is commercially available or prepared using methods available in the literature, is treated with a base, such as potassium carbonate, and (2S)-(+)-glycidyl-3-nitrobenzenesulfonate in a polar aprotic solvent, such as 2-butanone, at reflux. Standard purification procedures afford the epoxide 4a. 
A class of compounds of Formula(IIb) that are functionally equivalent to epoxides but contain no 3-membered oxirane can be prepared by a variety of methods familiar to those skilled in the art. One route is shown in Scheme 4. This is substantially the same procedure described by E. J. Corey and J. O. Link, J. Org. Chem. , 56, 442, (1991). The methyl ketone 5, available commercially or readily prepared by methods described in the literature, is treated with copper(II) bromide or dioxane/bromine to give the xcex1-bromoketone 6. This is then asymmetrically reduced using chiral reducing agents, such as, (R) or (S)-Alpine borane or (R) or (S)-tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrolo[1,2-c][1,3,2]oxazaborole-borane to afford the chiral alcohol 7. This alcohol group can then be protected most conveniently by a silicon based reagent, such as, triethylsilyl chloride; or the bromine can be replaced by the more reactive iodine by treating compound 7 with sodium iodide. This iodo-hydrin 8 can be O-protected using the same procedure as is used for compound 7 to afford compounds of Formula (IIb). 
Compounds of Formula (III) have not yet been described in the literature and a method for their synthesis needed to be developed. This route is illustrated in Scheme 5. Several amines of type 9 are commercially available or readily prepared by one skilled in the art. The primary amine is protected selectively as a suitable carbamate derivative 10, with, for example, di-tert-butyl dicarbonate. The nitro functional group is reduced using catalytic hydrogenation conditions, such as 5% Pd/C and H2 gas, to provide the aniline 11. The primary aromatic amine group is transformed into a diazonium group by treatment with sodium nitrite in cold hydrochloric acid. The diazonium salt is not isolated but treated immediately with potassium ethyl xanthate in aqueous sodium bicarbonate to afford the aromatic xanthate 12. This xanthate 12 is then treated with sodium borohydride followed by an alkylating agent, such as, benzyl bromide to give the sulfide 13. This material may be treated with mCPBA to afford either the sulfoxide or the sulfone. One molar equivalent of mCPBA forms a sulfoxide 14; two molar equivalents of mCPBA form the sulfone 15. Deprotection of the amine with an acid, such as trifluoroacetic acid, affords the desired intermediates of Formula (III). 
Compounds of Formula (III) where R2 is methyl can be prepared from phenylalanine as illustrated in Scheme 6. It is possible to use one of the known methods of reducing an amino acid to an xcex1-methyl amine. See B. G. Donner, Tetrahedron Lett., 36(8), pp. 1223-1226; or J. D. Bloom, et. al., J. Med. Chem., 35, pp. 3081-3084 (1992). Preferably, we developed our own method for reducing the amino acid to xcex1-methyl amines. Following the reduction, nitration via electrophilic aromatic substitutions affords the aromatic nitro compound 16. See Advanced Organic Chemistry, b 4th Ed., J. March, John Wiley and Sons, New York, 1992. Then by further electrophilic aromatic substitution the R3 group is introduced into the position meta to the nitro group, 17a. If the R3 group is to be ortho to the nitro group, 17b, the nitro group is reduced and acylated, then treated with butyllithium followed by the electrophile R3-X to afford the desired compound. See V. Snieckus, Bull. Soc. Chim. France, 1988, Nxc2x0 1, pp. 67-78. Reduction of the nitro compound 17a or hydrolysis of the amide 17b gives the compound 11a, which can be carried on to compounds of Formula (III), as shown in Scheme 6.
Intermediates of Formulas (II) and (III) are coupled by heating them neat or as a solution in a polar solvent such as methanol, tetrahydrofuran, or dimethylsulfoxide for 6 to 72 hours at temperatures from 25 to 125xc2x0 C. This provides compounds of Formula (I) or a protected form of compounds of Formula (I). The reaction is conveniently carried out by heating Intermediates (II) and (III) in a mixture of methanol and THF at 50xc2x0 C. for 48 hours. This procedure is illustrated in Scheme 7.
If a deprotection step is necessary it can immediately follow the coupling step just described.
In the examples where ring B is a benzene ring fused to a heterocyclic ring a slight variation is necessary and is described in Scheme 8. 2-Amino-3-nitophenol is treated as described in Scheme 3 to afford the ether 18. This material is heated with an amine of Formula (III) as described in Scheme 7 to provide the compound of Formula (Id). Reduction of the nitro group using sodium dithionite followed by either: 1) treatment with sodium nitrite in acetic acid (according to the procedure of Fries et al., Justus Liebigs Ann. Chem., 511, (1934) 213-230), or 2) heating with a carboxylic acid (according to the procedure of Bugaut and Kalopisses, Ger. Patent DE1921911 700226) to afford compounds of Formula (Ie). 
wherein:
W is N, or C-R5 
R5 is as described previously under Formula (I)
Intermediates of Formulas (IIb) and (III) are coupled by heating them neat or as a solution in a polar solvent such as methanol, tetrahydrofuran, or dimethylsulfoxide for 6 to 72 hours at temperatures from 25 to 145 C. This provides a protected form of compounds of Formula (I). The reaction is conveniently carried out by heating Intermediates (II) and (III) in a mixture of THF and methanol in a sealed tube at 50 to 145xc2x0 C. for 12 to 48 hours. The silyl protecting group is removed from compound of Formula (Ib) by treatment with TBAF to afford final compound of Formula (I). Other protecting groups would be removed by procedures known by those skilled in the art. This procedure is illustrated in Scheme 9.
Compounds of the general Formula (I) may be separated into diastereomeric pairs of enantiomers by, for example, fractional crystallization from a suitable solvent such as methanol or acetonitrile or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual optical stereoisomers by conventional means, such as, by the use of an enantiomerically pure chiral organic acid as a resolving agent. Alternatively, any enantiomer of a compound of Formula (I) may be obtained by stereospecific synthesis using optically pure starting materials of known configuration for the preparation of intermediates.
The compounds of this invention are useful in treating metabolic disorders related to insulin resistance or hyperglycemia, typically associated with obesity or glucose intolerance. The compounds of this invention are therefore, particularly useful in the treatment or inhibition of type II diabetes. The compounds of this invention are also useful in modulating glucose levels in disorders such as type I diabetes.
The ability of compounds of this invention to treat or inhibit disorders related to insulin resistance or hyperglycemia was established with representative compounds of this invention in the following standard pharmacological test procedures, which measured the binding selectivity to the xcex21, xcex22, and xcex23 adrenergic receptors. Binding to the receptors was measured in Chinese Hamster ovary (CHO) cells that were transfected with adrenergic receptors. The following briefly summarizes the procedures used and results obtained.
Transfection of CHO cells with xcex21 and xcex22 adrenergic receptors: CHO cells were transfected with human xcex21 or xcex22-adrenergic receptors as described in Tate, K. M., Eur. J. Biochem., 196:357-361 (1991).
Cloning of Human xcex23-AR Genomic DNA: cDNA was constructed by ligating four polymerase chain reaction (PCR) products using the following primers: an ATG-Narl fragment, sense primer 5xe2x80x2-CTTCCCTACCGCCCCACGCGCGATC3xe2x80x2 and anti-sense primer 5xe2x80x2CTGGCGCCCAACGGCCAGTGGCCAGTC3xe2x80x2; a Narl-Accl fragment, 5xe2x80x2TTGGCGCTGATGGCCACTGGCCGTTTG3xe2x80x2 as sense and 5xe2x80x2GCGCGTAGACGAAGAGCATCACGAG3xe2x80x2 as anti-sense primer; an Accli-Styl fragment, sense primer 5xe2x80x2CTCGTGATGCTCTTCGTCTCACGCGC3xe2x80x2 and anti-sense primer 5xe2x80x2GTGAAGGTGCCCATGATGAGACCCAAGG3xe2x80x2 and a Styl-TAG fragment, with sense primer 5xe2x80x2CCCTGTGCACCTTGGGTCTCATCATGG3xe2x80x2 and anti-sense primer 5xe2x80x2CCTCTGCCCCGGTTACCTACCC3xe2x80x2. The corresponding primer sequences are described in Mantzoros, C. S., et. al., Diabetes 45: 909-914 (1996). The four fragments are ligated into a pUC 18 plasmid (Gibco-BRL) and sequenced. Full length xcex23 AR clones (402 amino acids) containing the last 6 amino acids of hxcex23xe2x80x94AR are prepared with the xcex23-xcex2ARpcDNA3 from ATTC.
Binding Procedure: Clones expressing receptor levels of 70 to 110 fmoles/mg protein were used in the test procedures. CHO cells were grown in 24-well tissue culture plates in Dulbecco""s Modified Eagle Media with 10% fetal bovine serum, MEM non-essential amino acids, Penicillin-Streptompycin and Geneticin. On the day of test procedure, growth medium was replaced with preincubation media (Dulbecco""s Modified Eagle Media and incubated for 30 minutes at 37xc2x0 C. Preincubation medium was replaced with 0.2 ml treatment medium containing DMEM media containing 250 xcexcM IBMX (isobutyl-1-methylxantine) plus 1 mM ascorbic acid with test compound dissolved in DMSO. Test compounds were tested over a concentration range of 10xe2x88x929M to 10xe2x88x925M for xcex23 cells and 10xe2x88x928 to 10xe2x88x924M for xcex21 and xcex22 transfected cells. Isoproterenol (10xe2x88x925M) was used as an internal standard for comparison of activity. Cells were incubated at 37xc2x0 C. on a rocker for 30 min with the xcex23 cells and 15 min for xcex21 and xcex22 cells. Incubation was stopped with the addition of 0.2N HCl and neutralized with 2.5N NaOH. The plates, containing the cells and neutralized media, were stored at xe2x88x9220 degrees celsius until ready to test for cAMP using the SPA test kit (Amersham).
Data Analysis and Results: Data collected from the SPA test procedure were analyzed as per cent of the maximal isoproterenol response at 10xe2x88x925M. Activity curves were plotted using the SAS statistical and graphics software. EC50 values were generated for each compound and the maximal response (IA) developed for each compound is compared to the maximal response of isoproternol at 10xe2x88x925M from the following formula:
IA=% activity compound/% activity isoproterenol
Table I shows the xcex23-adronergic receptor EC50 and IA values for the representative compounds of this invention that were evaluated in this standard pharmacological test procedure. These results show that compounds of the present invention have activity at the xcex23-adrenergic receptor. The compounds of this invention had weaker or no activity at xcex21 and/or xcex22-adrenergic receptor.
Based on the results obtained in these standard pharmacological test procedures, representative compounds of this invention have been shown to be selective xcex23 adrenergic receptor agonists and are therefore useful in treating metabolic disorders related to insulin resistance or hyperglycemia (typically associated with obesity or glucose intolerance), atherosclerosis, gastrointestinal disorders, neurogenetic inflammation, glaucoma, ocular hypertension, and frequent urination; and are particularly useful in the treatment or inhibition of type II diabetes, and in modulating glucose levels in disorders such as type I diabetes. As used herein, the term modulating means maintaining glucose levels within clinically normal ranges.
As used in accordance with this invention, the term providing an effective amount means either directly administering such a compound of this invention, or administering a prodrug, derivative, or analog which will form an effective amount of the compound of this invention within the body.
It is understood that the effective dosage of the active compounds of this invention may vary depending upon the particular compound utilized, the mode of administration, the condition, and severity thereof, of the condition being treated, as well as the various physical factors related to the individual being treated. As used in accordance with invention, satisfactory results may be obtained when the compounds of this invention are administered to the individual in need at a daily dosage of from about 0.1 mg to about 1 mg per kilogram of body weight, preferably administered in divided doses two to six times per day, or in a sustained release form. For most large mammals, the total daily dosage is from about 3.5 mg to about 140 mg. It is preferred that the administration of one or more of the compounds herein begin at a low dose and be increased until the desired effects are achieved.
Such doses may be administered in any manner useful in directing the active compounds herein to the recipient""s bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, intranasally, vaginally, and transdermally. For the purposes of this disclosure, transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the present compounds, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).
Oral formulations containing the active compounds of this invention may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g. corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. Oral formulations herein may utilize standard delay or time release formulations to alter the absorption of the active compound(s).
In some cases it may be desirable to administer the compounds directly to the airways in the form of an aerosol.
The compounds of this invention may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparation contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository""s melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.
The compounds of the present invention also possess utility for increasing lean meat deposition and/or improving lean meat to fat ratio in edible animals, i.e. ungulate animals and poultry.
Animal feed compositions effective for increasing lean meat deposition and for improving lean meat to fat ratio in poultry, swine, sheep, goats, and cattle are generally prepared by mixing the compounds of the present invention with a sufficient amount of animal feed to provide from about 1 to 1000 ppm of the compound in the feed. Animal feed supplements can be prepared by admixing about 75% to 95% by weight of a compound of the present invention with about 5% to about 25% by weight of a suitable carrier or diluent. Carriers suitable for use to make up the feed supplement compositions include the following: alfalfa meal, soybean meal, cottonseed oil meal, linseed oil meal, sodium chloride, cornmeal, cane molasses, urea, bone meal, corncob meal and the like. The carrier promotes a uniform distribution of the active ingredients in the finished feed into which the supplement is blended. It thus performs an important function by ensuring proper distribution of the active ingredient throughout the feed. The supplement is used as a top dressing for the feed, it likewise helps to ensure uniformity of distribution of the active material across the top of the dressed feed.
The preferred medicated swine, cattle, sheep and goat feed generally contain from 0.01 to 400 grams of active ingredient per ton of feed, the optimum amount for these animals usually being about 50 to 300 grams per ton of feed. The preferred poultry and domestic pet feed usually contain about 0.01 to 400 grams and preferably 10 to 400 grams of active ingredient per ton of feed.
For parenteral administration the compounds of the present invention may be prepared in the form of a paste or a pellet and administered as an implant, usually under the skin of the head or ear of the animal in which increase in lean meat deposition and improvement in lean mean to fat ratio is sought. In general, parenteral administration involves injection of a sufficient amount of the compounds of the present invention to provide the animal with 0.001 to 100 mg/kg/day of body weight of the active ingredient. The preferred dosage for swine, cattle, sheep and goats is in the range of from 0.001 to 50 mg/kg/day of body weight of active ingredient; whereas, the preferred dose level for poultry and domestic pets is usually in the range of from 0.001 to 35 mg/kg/day of body weight.
Paste formulations can be prepared by dispersing the active compounds in a pharmaceutically acceptable oil such as peanut oil, sesame oil, corn oil or the like. Pellets containing an effective amount of the compounds of the present invention can be prepared by admixing the compounds of the present invention with a diluent such as carbowax, carnuba wax, and the like, and a lubricant, such as magnesium or calcium stearate, can be added to improve the pelleting process. It is, of course, recognized that more than one pellet may be administered to an animal to achieve the desired dose level which will provide the increase in lean meat deposition and improvement in lean meat to fat ratio desired. Moreover, it has been found that implants may also be made periodically during the animal treatment period in order to maintain the proper drug level in the animal""s body. For the poultry and swine raisers, using the method of the present invention yields leaner animals.
Additionally, the compounds of this invention are useful in increasing the lean mass to fat ratio in domestic pets, for the pet owner or veterinarian who wishes to increase leanness and trim unwanted fat from pet animals, the present invention provides the means by which this can be accomplished.